Antimicrobial Activities of Extracts and Isolated Coumarins from the Roots of Four Ferulago Species Growing in Turkey

Ferulago species have been utilized since ancient times as digestive, sedative, aphrodisiac, along with in salads or as a spice due to their special odors. The study reports isolation and characterization of bioactive compounds of Ferulago pachyloba (F. pachyloba), Ferulago trachycarpa (F. trachycarpa), Ferulago bracteata (F. bracteata), and Ferulago blancheana (F. blancheana) via bioassay guided fractionation and isolation process. The structures of compounds were elucidated by detailed spectroscopic analyses. They were also assessed for their activities at 1000-31.25 µg/mL concentrations by microbroth-dilution methods. Antimicrobial activity of aqueous, methanol extracts and dichloromethane, ethyl acetate, n-butanol and aqueous residue fractions of methanol extracts from aerial parts and roots of species along with isolated compounds [osthole, imperatorin, bergapten, prantschimgin, peucedanol-2′-benzoate, grandivitinol, suberosin, xanthotoxin, felamidin, marmesin, umbelliferone, ulopterol and a sterol mixture consisted of stigmasterol, β-sitosterol] were evaluated. Antimicrobial effect has been seen against Gram-negative, Gram-positive bacteria, and a yeast C. albicans at a concentration between 31.25 and 62.5 μg/mL. Especially, C. albicans (MIC = 31.25 μg/mL) was the most inhibited microorganism. Moreover, growth of P. aeruginosa, B. subtilis, E. coli, and S. aureus were inhibited at 62.5 μg/mL MIC values. Among tested samples prantschimgin and dichloromethane fraction of aerial parts from F. pachyloba showed the best activity against C. albicans (MIC = 31.25 μg/mL). However, among aqueous extracts and residue fractions, only F. blancheana aerial parts, F. trachycarpa aerial parts, and roots and F. bracteata roots showed activity against C. albicans. Among microorganisms E. coli was found to be the least affected.

resulted from mineral nutrient, supplying water status, temperature, and to insect pests. The composition of biologically active compounds from medicinal plants changes largely depending on the plant species, on their association with microbes and soil type. These bioactive secondary metabolites synthesized by medicinal plants can also extremely influence their physiological functions and plant-associated microbial communities (6).
Antimicrobial resistance has being increased rapidly against current drugs during the last decades; however, new antimicrobial drug development has slow down. This situation leads health authorities to search for natural antimicrobial active substances and/or to combine them with existing approved drugs. Treatment with plants is actually a traditional method known from antic ages long before the development of modern medicine (34,35). Antimicrobial activity of the plants comes from mostly by aromatic or phenolic substances (36). Antimicrobial substances of plants may be classified as alkaloids, essential oils, flavones, lectins, polyphenols, polypeptides, phenolics, tannins, and terpenoids (34). The current study aimed to give first report on evaluating antimicrobial activities of the extracts from root and aerial parts of F. pachyloba, F. bracteata, F. blancheana, F. trachycarpa, and isolated compounds.

Reagents and chemicals
Column chromatographies were performed on Silica gel 60 (0.063-0.200 mm, Merck) and Sephadex . TLC was carried out on pre-coated Kieselgel 60 F 254 aluminum sheets (Merck). Mueller Hinton Broth (MHB) (Merck) for the production of single-colony bacteria and Sabouraud Dextrose Agar (SDA) (Oxoid) for the production of single colony yeast for fresh culture and in macrodilution broth were used. Stock bacterial suspensions were prepared at a density of 0.5 McFarlandwith a DEN-1 densitometer (BIOSAN) device on physiological saline solution-from overnight cultures of standard strains.

Extraction and isolation
Air-dried roots and aerial parts of Ferulago pachyloba, F. trachycarpa, F. bracteata and F. blancheana were powdered and macerated three times with methanol for 8 h in a water bath not exceeding 45 °C (4 × 2 L) using a mechanical mixer at 300 rpm, separately. Combined extracts were filtered and concentrated till dryness by rotary evaporator (Heidolph VV2000, Germany) then dispersed in methanol-water (1:9) and fractionated four times with 400 mL of dichloromethane, ethyl acetate, and n-butanol, respectively. The fractions were concentrated till dryness by rotary evaporator. On the other hand, 50 g of roots and aerial parts from these plants were grounded and macerated with 500 mL of distilled water for 8 h/3 days at 30 to 35 °C, separately. Aqueous extract was filtered, freezed (Sanyo Medical Freezer, Germany), and lyophilized (Christ® Gamma 2-16 LSC, Germany) to give aqueous extracts of roots and aerial parts. Amounts of the powdered plants and obtained extracts are given in Table 1. A column of 52.5 cm in length and 6.9 cm in inner diameter was used in the column chromatography.
As a result of the bioguided fractionation study, the effective dichloromethane extracts of roots from all species were first submitted to a silica gel column and eluted with a gradient of n-hexane:ethyl acetate (100:0 → 0:100, v/v) and ethyl acetate:methanol (100:0 → 0:100, v/v), and nine fractions (Fr. A-I) were obtained. Fr. A was subjected to a silica gel column which was eluted with a mixture of n-hexane:ethyl acetate (95:5) and compounds 13 and 14 were obtained as a mixture. Repetitive silica gel column chromatography with n-hexane-ethyl acetate (90:10 and 95:5) solvent system on Fr. B gave compound 1. Fr. C was applied to silica gel column eluting with n-hexane:ethyl acetate (85:15) and Sephadex LH-20 column eluting with ethyl acetate to give compounds 2 and 3. Eluting with n-hexane-ethyl acetate (90:10) over silica gel column of Fr. D gave compound 4 and Fr. E gave compounds 5, 6, and 7. Fr. F eluted with 25% ethyl acetate in n-hexane and rechromatographed with 25% ethyl acetate in n-hexane on silica gel column to obtain compound 8. Fr. G was fractioned by column chromatography over silica gel using n-hexane:ethyl acetate mixtures (70:30 and 90:10) consecutively and compound 9 was obtained. Fr. H was submitted on a silica gel column using n-hexane:ethyl acetate (65:35) to yield compound 10 and the resulting fraction was chromatographed on silica gel column using n-hexane:ethyl acetate (90:10) to give compound 11. Fr. I gave compound 12. Compounds 1-4, 6, 8, 9, 11 and 13-14 were isolated by the same chromatographic methods in all species. Compounds 5, 10 and 12 were isolated only  Table 2.

Identification of isolated compounds
The structures of these isolated compounds were elucidated by means of detailed

Preparation of inoculum
Firstly, standard strains at -80 °C were inoculated to Mueller Hinton Broth (MHB, Merck) culture medium for bacteria and Sabouraud Dextrose Broth (SDB, Oxoid) for yeast and after 24 h of incubation (at 37 °C for bacteria and at 25 °C for Candida albicans) passages were made on Mueller Hinton Agar (MHA, Merck) medium and Sabouraud Dextrose Agar (SDA, Oxoid) to obtain single colony culture. They were left for 24 h incubation to obtain sufficient reproduction. Isolated colonies from fresh overnight culture of these strains were inoculated into physiological saline solution to turbidity compared to that of 0.5 McFarland standards. Then, 0.1 mL of these bacterial suspensions were transferred into tubes containing 20 mL of MHB for bacteria and SDB for yeast. This bacterial suspension was used in experiments.

Antimicrobial assay
Studies of the extracts, fractions, and isolated compounds were performed due to the standard reference methods for bacteria S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853, B. subtilis ATCC 6633 and for yeast C. albicans ATCC 10231. Minimum inhibitor concentration (MIC) was determined using the macrodilution broth method. The required     concentrations of the compounds were dissolved in DMSO (2%). One milliliter of extract was added in the first tube for each extract to be tested. Then, two fold 8 serial dilutions were made to give concentrations ranging from 1000 to 7.81 µg/mL. After adding 1 mL bacterial suspensions to the tubes, they were left for 18-24 h incubation. As a-negative control, only the bacterial suspension was added into 9 th tube containing 1 mL of broth media. At the end of the incubation period, the assessments were evaluated according to the turbidity of the tubes.

Preparation of inoculum
The tests were carried out according to the CLSI recommendations (37).

Statistical analysis
All the results are expressed as mean ± SE and the differences between means were statistically analyzed using one-way analysis of ANOVA followed by Bonferroni's complementary analysis, with P < 0.05 considered to indicate statistical significance (Figure 2).
The antimicrobial activities of the extracts, fractions, and isolated compounds have been given in Table 3 as MIC values. These compounds showed a broad range of (31.25-1000 µg/mL) antimicrobial activity. Among the lyophilized aqueous extracts only aerial parts of F. blancheana and also, among the aqueous residue fractions only aerial parts and roots of F. blancheana, F. trachycarpa, and roots of F. bracteata showed activity against C. albicans.
Thus, the structure of the compound 5 was characterized as peucedanol-2′-benzoate.   Our results were similar to previous studies of related coumarins. Karunai et al. (2012) found that ulopterol showed appreciable antimicrobial activity against some Gram negative and Gram positive microorganisms (49). Ojala et al. (2000) indicated that umbelliferone showed antibacterial activity against P. aeruginosa, bacteriostatic activity against E. coli; however, it did not show any activity against S. aureus, B. subtilis, and C. albicans (50). Golfakhrabadi et al. (2016) reported that prantschimgin had antimicrobial activity against S. aureus, P. aeruginosa, C. albicans, and also no activity against E. coli (32). Basile et al. (2009) showed that felamidin exposed antimicrobial activity against S. aureus and P. aeruginosa (8). It was reported that petroleum ether extracts of F. asparagifolia, F. aucheri, and chloroform extract of F. humilis which were collected from Aegean division of Turkey, did not show any significant activity by disc diffusion method against the tested microorganisms (49). Bostanlık et al. (2015) found that the extracts of Ferulago sandrasica Peşmen and Quezel and Ferulago mughlae Peşmen had antimicrobial activity against S. aureus ATCC 25923, but no activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and B. subtilis ATCC 6633 (51). Differences come from the difference of the compounds and their quantities among species. It is important to find a species that has a wide range of antimicrobial activity in the genus. Nowadays, due to the rapid increases in resistance to antibiotics, researches are shifting to create new combinations of active compounds derived from natural products. Besides, the consumers prefer foods with natural preservatives. As we mentioned before, these species except F. trachycarpa are endemic and this is the first report of their antimicrobial activity. We think that results of our study will contribute to the investigations in new antibiotic combinations or food preservatives. Therefore, based on our results and regarding the results of our colleagues, it seems that the biological activity assessed and sighted in the current study could be related to the synergistic effect of the different compounds included in these species. It is hoped that the research and development studies on the antimicrobial effects of plantderived compounds in relation to the use of current technological conditions, will broaden the scope of the solution field.
In conclusion, among the isolated compounds prantschimgin has emerged as new target for antimicrobial diseases. Therefore, we can conclude that prantschimgin can be used in antimicrobial diseases and may represent an herbal alternative to synthetic drugs.